Highly potent, naturally acquired human monoclonal antibodies against Pfs48/45 block Plasmodium falciparum transmission to mosquitoes

Summary Malaria transmission-blocking vaccines (TBVs) aim to induce antibodies that interrupt malaria parasite development in the mosquito, thereby blocking onward transmission, and provide a much-needed tool for malaria control and elimination. The parasite surface protein Pfs48/45 is a leading TBV candidate. Here, we isolated and characterized a panel of 81 human Pfs48/45-specific monoclonal antibodies (mAbs) from donors naturally exposed to Plasmodium parasites. Genetically diverse mAbs against each of the three domains (D1–D3) of Pfs48/45 were identified. The most potent mAbs targeted D1 and D3 and achieved >80% transmission-reducing activity in standard membrane-feeding assays, at 10 and 2 μg/mL, respectively. Co-crystal structures of D3 in complex with four different mAbs delineated two conserved protective epitopes. Altogether, these Pfs48/45-specific human mAbs provide important insight into protective and non-protective epitopes that can further our understanding of transmission and inform the design of refined malaria transmission-blocking vaccine candidates.


In brief
The malaria parasite surface protein Pfs48/45 is a leading transmissionblocking vaccine candidate, but little is known about the specificity of naturally acquired antibodies against this target. Fabra-García et al. isolate and characterize a panel of 81 human monoclonal antibodies from naturally exposed individuals and demonstrate that the most potent antibodies target domains 1 and 3 of Pfs48/45.

INTRODUCTION
Malaria is a devastating disease caused by Plasmodium parasites that are transmitted by Anopheles mosquitoes. Despite intensive malaria control efforts, the number of malaria cases and related deaths have increased in recent years. 1 Furthermore, the success of control programs is threatened by the emergence of artemisinin-resistant parasites in Africa 2 and the spreading of insecticide-resistant mosquitoes. 1 There is a broad consensus that novel tools, including tools that specifically block transmission, are needed to further reduce the burden of malaria and to continue progress toward malaria eradication.
Plasmodium transmission relies on the uptake of circulating gametocytes via a bloodmeal by female Anopheles mosquitoes. Inside the mosquito midgut, male and female gametocytes are activated and form gametes that egress from red blood cells to undergo fertilization. After fertilization, the parasites go through several developmental stages that lead to the formation of oocysts. Inside oocysts, sporozoites are formed that migrate to the salivary glands resulting in an infectious mosquito. Transmission-blocking vaccines (TBVs) aim to interrupt transmission from human to mosquito by inducing antibodies in humans that block parasite development inside the mosquito midgut and hence prevent onward transmission to other humans. 3,4 Pfs48/45 is a leading TBV candidate, present on the surface of late-stage gametocytes and activated gametes, and comprises three 6-cysteine domains and a glycosylphosphatidylinositol (GPI) anchor 5,6 ( Figure 1A). Parasites that lack Pfs48/ 45 fail to generate oocysts, and rodent malaria parasites without P48/45 produce infertile male gametes, strongly suggesting that Pfs48/45 plays a key role in gamete fertilization. 7 Pfs48/45-specific rodent monoclonal antibodies (mAbs), raised against whole parasites, can prevent oocyst formation 8-10this formed the basis for the development of Pfs48/45 as a TBV candidate. The most potent transmission-blocking mAb described to date is 85RF45.1, 10 which targets the conserved epitope I on Pfs48/45 domain 3 (D3, also known as Pfs48/ 45-6C) and blocks the transmission of genetically diverse Plasmodium falciparum (P. falciparum) strains. 11,12 A humanized version, TB31F, has recently been generated 11 and has completed early clinical evaluation. 13 The pre-clinical development of a Pfs48/45-based TBV has long been hampered by difficulties in producing correctly folded antigen (reviewed in Theisen et al. 14 ). However, there has been considerable progress recently with the expression of D3-containing constructs in Lactococcus lactis (R0.6C and ProC6C, where 6C denotes D3) [15][16][17] and full-length Pfs48/45 in Drosophila melanogaster S2 cells. 18 These vaccine candidates are currently being  6 Domain 3 is also known as ''6C'' and contains epitope I. 19 (B) Recognition of full-length Pfs48/45 by plasma samples from two naturally exposed donors in an enzyme-linked immunosorbent assay. Values are means of two technical replicates and error bars represent the SEM. Transmission-reducing activity (TRA) of purified total IgG, tested at 1:3 dilution in the presence of complement, from both donors are shown in the legend and are outcomes of two independent standard membrane-feeding assays. The raw SMFA data are included in Table S7. (C) Gating strategy for antigen-specific sorting. Pfs48/45-specific B cells were isolated by gating CD19+, IgG+, and Pfs48/45+ cells from single live lymphocytes. (D) Schematic representation of multiplexed bead-based (left) and soluble antigen (right) screening assays on the microfluidic device. Memory B cells were activated in vitro into antibody-secreting cells and Pfs48/45 reactivity was determined using either antigen-coated beads and a fluorescently labeled secondary antibody (left) or anti-human IgG capture beads with fluorescently labeled Pfs48/45 antigens (right). (E) Summary of domain specificity of isolated mAbs (Table S1). Unk, unknown domain specificity. (F) Pie charts showing the number of isolated Pfs48/45-specific mAbs per donor (center) and fractions of D1-, D2-, and D3-specific mAbs in yellow, red, and blue. mAbs with unknown domain specificity are shown in gray. See also Figures S1-S3 and Table S1. evaluated in phase I clinical trials (Clinicaltrials.gov IDs: NCT04862416, NCT05400746).
Pfs48/45 is expressed on the surface of gametocytes that, although located inside red blood cells, are circulating in the human bloodstream. The clearance of gametocyte-infected red blood cells by the spleen can expose this antigen to the human immune system, resulting in the natural acquisition of antibodies in gametocyte-carrying individuals (reviewed in Stone et al. 20 ). We recently purified Pfs48/45-specific polyclonal antibodies from naturally exposed donors and demonstrated their ability to block transmission of cultured gametocytes in the standard membrane-feeding assay (SMFA). 21 Together, these data demonstrate that Pfs48/45 is immunogenic and can induce naturally acquired functional antibodies in Plasmodium-infected humans, but little is known about the mAbs that make up the response. These mAbs could provide valuable insights into functional and non-functional epitopes and thereby inform vaccine design. Furthermore, potent human mAbs may also be considered for passive immunization strategies. 13,[22][23][24] Here, we isolated human Pfs48/45-specific mAbs from memory B cells (MBCs) of two naturally exposed donors with high serum transmission-reducing activity (TRA). We determined the domain and epitope specificity of these mAbs and linked these to functional activity. Finally, we used X-ray crystallography to delineate epitopes of potent D3-specific mAbs, providing atomic insights into mAb functional activity.

RESULTS
Isolation of Pfs48/45-specific MBCs from naturally exposed donors For the isolation of Pfs48/45-specific mAbs, we selected two donors that had experienced repeated Plasmodium infections. One donor was a 69-year-old Dutch expatriate who had lived in central Africa for approximately 30 years. This donor had total IgG that recognized full-length Pfs48/45 in ELISA and strongly reduced transmission ( Figure 1B; donor A in Stone et al. 21 ). We selected the other donor from a panel of 1,358 donors from Tororo, an area with high malaria transmission in Uganda with an estimated exposure of 310 P. falciparum-infected mosquito bites per person per year. 25 Plasma samples from these donors were screened for (1) high antibody titers against gametocyte extract, (2) high TRA of purified IgGs in SMFA, and (3) the presence of antibodies against Pfs48/45. The total IgG of the selected 8-year-old Ugandan donor showed 100% TRA in SMFA and recognized full-length Pfs48/45 by ELISA, albeit to a lesser extent than that of the Dutch expatriate ( Figure 1B). Using fluorescently labeled full-length Pfs48/45, we sorted 123 single MBCs from the Dutch expatriate and obtained 46 unique paired antibody sequences ( Figure 1C). We also used a microfluidic device to screen single MBCs from both donors for Pfs48/45 reactivity ( Figure 1D). This screening method identified 601 Pfs48/45-specific hits from which 91 unique paired antibody sequences were obtained. We recombinantly expressed 100 unique antibodies, obtained from one or both of the B cell screening methods, as human IgG1 to confirm specificity to Pfs48/45. Eighty-one mAbs bound to full-length recombinant Pfs48/45 in ELISA (Table S1) or showed high-affinity binding by surface plasmon resonance (SPR) ( Figure S1A), and 74 of these recognized Pfs48/45 in gametocyte extract by western blot (Figure S2A). All 81 mAbs recognized Pfs48/45 in its native configuration on the female gamete surface membrane as detected by surface immunofluorescence assay ( Figure S2B). Altogether, we isolated 81 Pfs48/45-specific mAbs, which recognized the surface of female gametes, from two naturally exposed donors.

Isolated mAbs target different domains of Pfs48/45
To determine the domain specificity of Pfs48/45-specific mAbs, we produced three Pfs48/45 protein fragments that contained D1-2, D2-3, and D3 only, respectively (Figures S1B and S1C). Previously described rodent mAbs 10 recognized these fragments in western blot and ELISA, confirming that the fragments contained domains that were properly folded (Figures S1D and S1E). Using these protein fragments, we tested the binding of the 81 human mAbs in ELISA and found that 22 antibodies bound to D1, 36 bound to D2, and 15 bound to D3 ( Figure 1E; Table S1). Seven mAbs did not show reactivity with any of the fragments. One mAb, RUPA-154, reacted with all three constructs, suggesting it targets an epitope that spans multiple domains. Eleven out of 22 D1-specific mAbs bound to native Pfs48/45 protein under reducing conditions and thus target an epitope that is primarily linear ( Figure S2A). The other mAbs, including all D2-and D3specific antibodies, lacked reactivity under reducing conditions and therefore target more conformational epitopes ( Figure S2A). Having established domain specificity, we next mapped the fine specificity of the human mAbs in competition experiments, which included previously described rodent mAbs 10,26 and the highly potent humanized mAb TB31F 11 as reference mAbs. mAbs fell into 42 bins that could be grouped into four larger clusters ( Figure S3A). The first cluster contains previously identified D1-specific rat mAb 85RF45.5, the second contains D2-specific rat mAb 85RF45.3, the third contains D3-specific rat mAb 85RF45.1, mouse mAb 32F3, and the humanized mAb TB31F, whereas the fourth lacks reference mAbs. Competition patterns within clusters are complex and clusters show extensive interactions with each other, suggesting a high diversity in epitope specificity ( Figure S3A). The clusters defined by competition analyses align well with the domain specificity determined by ELISA ( Figure S3B). Some D1-specific mAbs compete with D3specific mAbs, suggesting that D1 and D3 may be in close relative proximity to each other in the full-length protein, or that the epitopes may be allosterically interconnected. Altogether, we identified 81 human mAbs that cover a range of specificities: 78 were obtained from the Dutch expatriate and 3 were obtained from the Ugandan donor ( Figure 1F), consistent with the difference in observed antibody titers in plasma.

Potent transmission-blocking mAbs bind Pfs48/45-D1 and D3
To determine the functional potency of the human mAbs, we tested these in a series of membrane-feeding assays with cultured P. falciparum NF54 gametocytes and Anopheles stephensi mosquitoes. We first screened the mAbs at 100 mg/mL in a barcoded membrane-feeding assay that quantifies the percentage of low-infected mosquitoes to identify mAbs with strong TRA. 27 In this assay, the mAbs displayed a wide range of activities-the most potent mAbs target D1 and D3, whereas most of the D2-specific mAbs showed weak TRA (Figure 2A). To confirm high potency, we next tested the 36 most potent mAbs in SMFA. We also included five mAbs that were not yet available at the time of the high-throughput membrane-feeding assay. Twenty-seven mAbs showed more than 80% reduction in oocyst intensity at 100 mg/mL, including 12 D1-, two D2-, and 12 D3specific mAbs and one mAb with unknown domain specificity ( Figure S3D). This represents 55% (12/22), 6% (2/36), and 80% (12/15) of all unique D1-, D2-, and D3-specific mAbs, (A) mAbs were tested at 100 mg/mL in a barcoded membrane-feeding assay using Anopheles stephensi mosquitoes and transgenic Plasmodium falciparum NF54 parasites that express a luciferase reporter. 27 The figure shows the proportion of mosquitoes with low infection (>90% reduction in oocyst intensity relative to the vehicle controls) 8 days after feeding. Note that five mAbs, including RUPA-160, were not available at the time the barcoded membrane-feeding assay was performed and were only tested in a standard membrane-feeding assay ( Figure S3D). (B-D) D1-specific (B), D2-specific (C), and D3-specific (D) mAbs that showed >95% TRA in standard membrane-feeding assay (SMFA) at 100 mg/mL ( Figure S3D) were titrated to determine their potency. (E) The most potent D3-specific mAbs were further titrated and tested head-to-head with TB31F in the single SMFA experiment. mAbs are colored according to domain specificity and TRA values (B-E) were based on single SMFA experiments with 20 mosquitoes per condition and calculated as the percentage reduction in oocyst intensity compared with a negative control. Raw SMFA data and 95% confidence intervals are presented in Table S7. See also Figure S3. Article respectively. We then titrated mAbs that showed ˃95% TRA at 100 mg/mL to determine their potency in more detail ( Figures 2B-2D). D1-specific mAbs, except RUPA-58, were similarly potent with IC 80 values between 2 and 10 mg/mL, D2-specific RUPA-160 showed approximately 80% TRA at 10 mg/mL, whereas D3-specific mAbs showed a larger range of potencies. Four D3-specific mAbs, RUPA-29, -50, -54, and -100, showed more than 80% TRA at a low concentration of 2 mg/mL, similar to that of the most potent transmission-blocking antibody described to date, TB31F ( Figure 2E). Together, these data demonstrate that natural Plasmodium infection can induce functional antibodies against all three domains of Pfs48/45, that strong functional activity is observed for mAbs targeting both D1 and D3, and that the most potent mAbs target D3.  Table S2). The mAbs isolated from the Ugandan donor were genetically distinct from those acquired from the Dutch expatriate donor (Table S2). The two most expanded VH families were IGHV1-8 and IGHV4-34, comprising 26 mAbs that target D2 and nine mAbs that target D1, respectively ( Figures 3D and 3E). One of these families, IGHV1-8, contained the only two D2-specific mAbs (RUPA-25 and RUPA-160) with high potency in SMFA (i.e., >80% TRA at 100 mg/mL; Figure S3D). Although these mAbs share the same heavy-and light-chain gene segments with many other mAbs from this family, the complementarity-determining region 3 (CDR3) sequences are different, suggesting that the CDR sequences determine functional TRA. Although the majority of high potency mAbs that target D1 were genetically similar and contained VH4-34 segments, potent mAbs that target D3 were more genetically diverse. Although the heavy chains of the four most potent D3 antibodies (RUPA-29, RUPA-50, RUPA-54, and RUPA-100) are encoded by the related IGHV3-30 and IGHV3-33 germline genes, the light chains of these antibodies are encoded by the same lambda chain variable domain. Somatic hypermutations were generally low and similar across domain specificities ( Figure 3H). Potent mAbs did not contain more somatic hypermutations than other mAbs (p = 0.58) ( Figure 3I). The binding affinity of the mAbs ranges from the low nanomolar to micromolar range ( Figures 3J and S1A). Although we did not obtain affinity data for all mAbs, high potency mAbs did not have higher affinities nor lower off-or higher on-rates ( Figures 3J and S1). Together, these data demonstrate that the isolated mAbs are genetically diverse and that potency is not only determined by genetic origin, affinity maturation, or binding affinity.  Table S3; PDB: 7UNB). 29 First, to structurally characterize this epitope I in more detail, we examined the co-crystal structures of two potent antibodies that compete with TB31F, RUPA-29 (TRA of >80% at 2 mg/mL), and RUPA-47 (TRA of 100% at 100 mg/mL that drops to 20% at 10 mg/mL), as Fabs bound to D3 ( Figures 4A, 4B, S3C, and S4A-S4D; Table S3; PDB: 7UNB). RUPA-29 is one of several genetically similar antibodies with >80% TRA at 2 mg/mL, whereas RUPA-47 is highly inhibitory but less potent than some of the other D3 binders, making both antibodies informative for delineating epitope 1 (Figures 2D and 3A).
RUPA-29 binds primarily to b strands d and d 0 (residues 347-356), b strand d 00 (residues 368-371), and the intervening loop between b strands g and h (residues 413-416) of D3. Its interactions with D3 are mediated by all three CDRs of both the heavy chain (buried surface area [BSA] = 382 Å 2 ) and the light chain (BSA = 312 Å 2 , total BSA = 694 Å 2 ) ( Table S4). The light-chain forms four H-bonds with D3 mediated by antibody residues K 30 , Y 34 , Y 49 , and S 66 , and two salt bridges mediated by residues D 50 and D 51 ( Figure 4C). The heavy chain provides an additional four H-bonds from heavy-chain CDR1 (HCDR1) and HCDR2, and van der Waals interactions formed by HCDR3 residues including F 96 (54 Å 2 ), H 98 (68 Å 2 ), and F 100A (47 Å 2 ) (Table S4). RUPA-29, along with RUPA-100, RUPA-54, and RUPA-50, is part of a highly potent antibody lineage made up of an IGHV3-33 or IGHV3-30 heavy chain and an IGLV3-10 lambda chain. An alignment of the lambda-chain CDRs (LCDRs) of these four mAbs revealed that most of the RUPA-29 contact residues in the RUPA-29-D3 structure, including those that form electrostatic interactions with D3, are well conserved (Figures S5A-S5D; Table S4). Although the HCDR residues involved in RUPA-29 binding to D3 are more variable across this antibody lineage, these differences mainly occur at residues involved in van der Waals interactions that can be mediated by several different amino acids (Figures S5A-S5D; Table S4).   (Table S4). Both the heavy chain and light chain contribute to binding, with BSAs of 557 and 448 Å 2 , respectively. RUPA-47's heavy chain interacts extensively with loop 357-369 of Pfs48/45-D3 through H-bonds and salt bridges formed by HCDR1 and HCDR3 residues N 31 , R 94 , G 101 , and Y 102 ( Figure 4D). Kappa-chain CDR1 (KCDR1) and KCDR2 residues R 29 , Y 32 , and S 52 of the RUPA-47 light-chain form a salt bridge and several H-bonds with D 351 , Q 355 , and K 413 of Pfs48/45-D3 (Table S4).
An overlay of the RUPA-47-bound and RUPA-29-bound D3 structures with the TB31F-bound D3 structure reveals that their epitopes overlap considerably with one another (Figures 4E and  4F). This finding is consistent with both RUPA-47 and RUPA-29 competing with TB31F in binding competition assays (Figure S3C). RUPA-29 and TB31F share the bulk of their key contacts on D3 and their variable domains are positioned similarly with regard to D3. Pfs48/45 residues that form salt bridges and H-bond with RUPA-29 and TB31F are largely shared (D 351 , Q 355 , Y 371 , K 413 , and K 416 ) ( Figure 4F). RUPA-47 and TB31F bind overlapping but slightly different sites, with RUPA-47 interacting more heavily with loop 357-369 of D3 and having a slightly different angle of approach (Figures 4E and 4F). All three antibodies bind D3 with nanomolar binding affinities, with K D 's of 3.7, 0.4, and 0.3 nM for TB31F, 11 RUPA-29, and RUPA-47, respectively (Table S5). Given that these are all strong binders to the recombinant protein, the lower inhibitory potency of RUPA-47 compared with TB31F and RUPA-29 may result from its different angle of approach or its epitope footprint being shifted toward loop 357-369 of D3. Overall, we structurally delineate this potent epitope bin as Ia.
Twelve non-synonymous single-nucleotide polymorphisms (SNPs) with varying frequencies have been identified in D3 across P. falciparum isolates. 30 These include V304I/D, L314I, D320H, S322N, P359A, I376L, A387T, K414N, K416N, T422K, and T436I. Out of these SNPs, three occur in epitope Ia (S322N, K414N, and K416N) ( Figure 4G). The S322N mutation is relatively common, with a frequency of 39.0%, whereas both K414N and K416N mutations are very rare, with frequencies of 0.007% and 0.03%, respectively. 30 RUPA-47 does not interact with any of these residues, whereas RUPA-29 forms a salt bridge and two H-bonds with K 416 through light-chain residues D 51 , S 66 , and K 30 . Kinetics experiments of Fabs RUPA-47 and RUPA-29 binding to a D3 construct containing the K416N mutation showed that RUPA-47's binding affinity remains unchanged, whereas RUPA-29's binding affinity drops with the introduction of this rare polymorphism but remains in the nanomolar affinity range (109 nM; Figure 4H; Table S5). Together, these results indicate that the epitope Ia antigenic site on D3 is largely conserved and can be recognized by potent human antibodies resilient to SNPs reported in field isolates.
Structural delineation of potent epitope Ib on Pfs48/ 45-D3 Using X-ray crystallography, we next uncovered the epitopes of non-TB31F-competing, potent antibodies RUPA-44 and RUPA-117 ( Figures 5A, 5B, and S4A-S4D; Table S3, PDB: 7UNB). 29 RUPA-44 and RUPA-117 have almost identical sequences, with just two amino acid substitutions in the light chain and four in the heavy chain ( Figure S5E). As a result, they bind to very similar epitopes and share the majority of contacts. RUPA-44 has a BSA of 841 Å 2 , with the heavy chain and the light chain contributing 544 and 297 Å 2 , respectively, whereas RUPA-117 has a BSA of 795 Å 2 , with the heavy chain and the light chain contributing 522 and 273 Å 2 , respectively (Table S6). Most of the interactions between these two antibodies and D3 are mediated by their HCDR3 loop (RUPA-117 = 402 Å 2 , RUPA-44 = 414 Å 2 BSA). The 19-residue HCDR3 of the antibodies forms a b-hairpin that interacts with b strand b of the D3 b sandwich (residues 324-331) ( Figure 5C). Both RUPA-44 and RUPA-117 have the same HCDR3 sequence. HCDR3 residues R 94 , M 100A , K 100B , V 100D , and I 100F form H-bonds and salt bridges with D3 residues D 320 , D 312 , H 324 , S 326 , and N 328 ( Figure 5C). The light-chain KCDR1 contributes additional H-bonds mediated by S 30 and S 31 for RUPA-44, and S 28 , S 30 , and I 31 for RUPA-117 ( Figure 5D). Despite these antibodies competing minimally with epitope Ia binders ( Figure S3C), there is a slight overlap between epitope Ia and epitope Ib. Indeed, five residues in RUPA-117's epitope (D 321 , S 322 , E 362 , E 363 , and K 416 ) and five residues in RUPA-44's epitope (D 321 , S 322 , E 362 , E 363 , and L 364 ) are also part of epitope Ia.
The epitope Ib is relatively conserved in P. falciparum isolates, with only four reported SNPs (L314I, D320H, S322N, and K416N) of 28.2%, 0.007%, 39.0%, 0.03% frequencies, respectively (Figure 5E). 30 Isolates containing both the S322N and L314I SNPs have also been found with a frequency of 1.1%. 30 RUPA-44 and RUPA-117 make van der Waals interactions with L 314 and S 322 . Both also form a salt bridge with D 320 and a H-bond with its backbone amide. Additionally, RUPA-117 also forms van der Waals interactions with K 416 . Binding kinetics experiments with D3 constructs containing the single L314I, D320H, S322N, and K416N point mutations revealed that none of these mutations substantially impact RUPA-44 or RUPA-117 bindingboth Fabs bound all antigens with affinities <20 nM ( Figure 5F; Table S5). We therefore structurally delineate an antigenic site on D3, which can be recognized by potent human antibodies that can accommodate known SNPs.

Article DISCUSSION
Here, we isolated highly potent human Pfs48/45 mAbs from individuals naturally exposed to Plasmodium. We characterized the binding and epitope specificity of 81 mAbs and linked these to functional activity, providing insight into functional and non-functional human antibody responses toward this leading TBV candidate.
Naturally acquired TRA occurs after exposure to circulating Plasmodium gametocytes that are not taken up by mosquitoes but cleared by the spleen. Strong functional TRA is a rare phenotype that is not predicted by cumulative exposure or increasing age and remains poorly characterized. 21 Several studies have demonstrated the presence of naturally acquired antibodies to Pfs48/45 in humans and its association with TRA (reviewed in Stone et al. 20 ). We recently demonstrated causality for this association-affinity-purified naturally acquired antibodies to D2 and D3 blocked transmission in SMFA. 21 Here, we provide insight into mAbs that make up polyclonal responses in humans and identified mAbs against all three domains of Pfs48/45. Many of these mAbs showed weak or negligible activity, particularly those that bind to D2. However, we identified 26 potent mAbs with more than 80% TRA at 100 mg/mL that mostly targeted D1 and D3. A similar pattern has been observed for rodent mAbs against Pfs48/45. 10,18,31 Potent mAbs did not have more somatic hypermutations or higher affinity, further supporting the hypothesis that the target epitope is the main determinant of functional activity.
Since the most potent mAbs bound to D3, many with 80%-100% TRA at or below 2 mg/mL, we characterized their interactions at a molecular level. Our epitope binning experiments revealed two protective epitopes on D3-potent antibodies competed with either TB31F, RUPA-117, or both. Structures of Pfs48/45-D3-Fab complexes were solved using X-ray crystallography to delineate the epitopes of four antibodies (RUPA-44, RUPA-117, RUPA-47, and RUPA-29). The structures of TB31F-competing human antibodies RUPA-29 and RUPA-47 allowed for a more detailed look at the Pfs48/45 epitope historically referred to as epitope I. A relatively wide range of TRA observed for the TB31F-competing antibodies suggests that the now-expanded epitope bin Ia antigenic site may be associated with subtle differences in antibody recognition that considerably impact potency, the molecular basis of which still needs to be fully uncovered. Out of the antibodies isolated, many share genetic similarities. RUPA-29 is one of several potent mAbs that have an IGHV3-33 or IGHV3-30 heavy chain paired with an IGLV3-10 lambda chain (RUPA-29, RUPA-100, RUPA-54, and RUPA-50). An alignment of the heavy chains and lambda chains of RUPA-100, RUPA-50, and RUPA-54 with RUPA-29 revealed that most of the lambda chain contact residues in the RUPA-29-Pfs48/45-D3 structure are shared among these anti-bodies, whereas the heavy-chain contacts are more variable (Figures S5A-S5D). Whether a germline-targeting approach for next-generation Pfs48/45 immunogen design can rely on preferentially re-eliciting antibodies with such genetic signatures to enhance the potency of the transmission-blocking responsean approach recently employed for vaccine development in other fields 32-34 -is an area of future exploration. Importantly, solving the crystal structures of potent human antibodies RUPA-44 and RUPA-117 bound to Pfs48/45-D3 allowed us to describe a potent Pfs48/45 epitope, epitope Ib, in atomic detail. This binding site, like epitope Ia, is highly conserved in P. falciparum with only four reported SNPs, none of which individually impacted either RUPA-44 or RUPA-117 binding. Mapping the binding sites of all Pfs48/45 antibodies currently characterized at the molecular level revealed that most potent antibodies against Pfs48/45-D3 bind to one face ( Figure 6). This could indicate that in the context of the native parasite, the opposite side of Pfs48/45-D3 may be inaccessible, potentially due to inter-domain contacts within Pfs48/45, interactions with other binding partners, or other in vivo considerations. Structural studies of Pfs48/45 in its native context will help evaluate this hypothesis.
Some mAbs against D1 and D3 competed with each other, suggesting that these domains may be in close proximity, which is supported by recent X-ray crystallography structures of fulllength Pfs48/45 in the context of mouse-derived antibodies of varying potencies. 28 An overlay of the co-crystal structures of D3 bound to RUPA-29, RUPA-44, RUPA-117, and RUPA-47 with the full-length crystal structure of Pfs48/45 28 indicates that these two potent epitopes are accessible in the full-length Pfs48/45 structure, as would be expected ( Figure S4E).
In this study, we have only screened the activity of individual human mAbs to Pfs48/45. Future studies may test combinations of mAbs to determine whether mAbs can act synergistically, as previously suggested. 26 These studies with combinations of mAbs should include RUPA-154, which may target an epitope that spans multiple domains and may be interesting in terms of eliciting additive effectiveness. Furthermore, it will be particularly interesting to determine whether D2-specific mAbs, which appear to be abundant and generally exhibit weak activity, can potentiate mAbs against the other domains or decrease their activity by, for instance, competition. If D2-specific mAbs decrease the activity of mAbs to other domains, this has important implications for the domains that should be included in Pfs48/45 TBV designs. These analyses on mAb potentiation or inhibition should not be restricted to Pfs48/45-specific mAbs but may also include mAbs against other TBV candidates such as Pfs230, as synergy between mAbs against different TBV candidates has been found previously. 35 In terms of future applicability, the mAbs identified in this study may be valuable for the development of passive and active immunization strategies to reduce malaria transmission. Four mAbs (RUPA-29, -50, -54, and -100) have potencies similar to TB31F, the most potent transmission-blocking mAb described to date. Clinical evaluation of TB31F in humans demonstrated that a single administration can achieve strong TRA for approximately 5 months, an effective time window that would cover peak transmission season in certain Sahelian areas. 13 The potent mAbs identified in this study may be relevant alternatives or additions to TB31F as they are of human origin and may be less likely to induce anti-drug antibodies. The identification of potent, naturally acquired human mAbs against Pfs48/45 also supports its further development as a TBV. Although clinical evaluation of R0.6C, containing Pfs48/45-D3, is ongoing (Clinicaltrials.gov ID: NCT04862416), our data suggest that the design(s) of next-generation Pfs48/45 vaccines should focus on epitopes Ia and Ib on D3. Future vaccine constructs may also include regions in D1 that are the target of potent mAbs but for which the exact identity still needs to be identified. D2 may be excluded from Pfs48/45-based vaccines as it seemed immunodominant in the Dutch expatriate donor and induced many mAbs with weak potency. The current work thus provides insights into protective and non-protective epitopes that can inform the design of next-generation constructs for this promising TBV antigen and may also form a starting point for effective passive immunization to reduce the transmission of P. falciparum.
Limitations of the study Our mAbs were obtained from a limited set of two genetically distinct donors with markedly different ages and infection histories. The majority of mAbs, 78, were obtained from a Dutch expatriate who was first exposed to Plasmodium at an adult age-only three mAbs were obtained from a Ugandan child who was selected from a large cohort of donors. There were no common germline precursors for mAbs from both donors. Nevertheless, mAbs from more donors would be required to fully capture the diversity of Pfs48/45 responses in different individuals and populations. Earlier studies have indeed suggested that antibody responses to Pfs48/45 differ between individuals. 36 Furthermore, we used full-length Pfs48/45 produced in insect cells to identify D1-and D2-reactive B cells. Our method using this recombinant glycoprotein ( Figure S1C) may, therefore, have failed to identify B cells directed to epitopes that are not glycosylated or differ in glycoforms in native Pfs48/45. Although our structural studies provide molecular detail on the epitopes of functional mAbs against D3, we do not know the exact target epitopes of D1-and D2-specific mAbs nor that of non-functional D3-specific mAbs. Identification of the target epitopes of these mAbs and structural studies on full-length Pfs48/45 with these mAbs will be an important area for future research.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

ACKNOWLEDGMENTS
We would like to thank Jonathan Mandel for assistance with ELISAs, Rianne Stoter and Wiebe Kooijman for parasite culture, Laura Pelser, Astrid Pouwelsen, Jacqueline Kuhnen, Jolanda Klaassen, and Saskia Mulder for mosquito dissection, Greg Wasney for assistance with biolayer interferometry (BLI), and Yimin Wu for the critical review of this manuscript. We are also grateful to Michael Theisen and Susheel Singh for providing the R0.6C construct.

AUTHOR CONTRIBUTIONS
The experimental design was collaborative between all co-authors. Experiments were conducted by A.

DECLARATION OF INTERESTS
The authors declare no competing interests.